Five years ago, the protein prestin (SLC26A5) was identified as the motor protein that drives electromechanical transduction in cochlear outer hair cells. Subsequent experiments have confirmed prestin is essential for both outer hair cell (OHC) electromotility and normal auditory function. This unique polytopic membrane protein contributes to the voltage sensor that detects changes in the transmembrane potential and to the motor mechanism of OHC electromotility. Prestin is a member of the SLC26A family of anion transporters which play critical roles in ion and fluid homeostasis as well as pH and cell volume regulation. The molecular basis of prestin motor function is presently unknown. The objective of this proposal is to advance our understanding of prestin function. In particular, we will test the hypothesis that prestin molecules self-associate to form functional complexes. The C-terminus of SLC26 family members contains a domain that is highly conserved throughout prokaryotes and eukaryotes, referred to as the STAS domain. Mutations in the STAS domain of several SLC26A proteins are responsible for a variety of human diseases including Pendred syndrome (SLC26A4), congenital chloride diarrhea (SLC26A3), and diastrophic dysplasia (SLC26A2). STAS domains from lower phyla have demonstrated interactions with the membrane. Based on these and other findings, we hypothesize that the STAS domain of prestin mediates prestin-prestin interactions and interactions between prestin and the membrane. Importantly, our preliminary data demonstrates that alterations in the membrane microenvironment alter prestin function. In this proposal, we will use biochemical, cellular, biophysical and optical (FRET and FRAP) approaches to probe the molecular interactions between prestin, prestin and the membrane, and the STAS domain and the membrane. Additionally, we will determine if SLC26A STAS domains can be functionally exchanged. The results from our studies will lead to a deeper understanding, not only of prestin function and the molecular basis of electromotility, but will also provide insights into the function of STAS domains from the SLC26A family. [unreadable] [unreadable] [unreadable]